Molecular determinants of chaperone interactions on MHC-I for folding and antigen repertoire selection Andrew C. McShana,1, Christine A. Devlinb,c,1, Sarah A. Overalla, Jihye Parkb,c, Jugmohit S. Toora, Danai Moschidia, David Flores-Solisa,2, Hannah Choib,c, Sarvind Tripathia, Erik Prockob,c,3, and Nikolaos G. Sgourakisa,3 aDepartment of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064; bDepartment of Biochemistry, University of Illinois, Urbana, IL 61801; and cCancer Center at Illinois, University of Illinois, Urbana, IL 61801 Edited by Peter Cresswell, Yale University, New Haven, CT, and approved November 8, 2019 (received for review September 9, 2019) The interplay between a highly polymorphic set of MHC-I alleles glucosyltransferase (UGGT) (3–5). The discovery that TAPBPR and molecular chaperones shapes the repertoire of peptide anti- can function as a peptide-exchange catalyst outside the peptide- gens displayed on the cell surface for T cell surveillance. Here, we loading complex and can maintain empty MHC-I molecules in a demonstrate that the molecular chaperone TAP-binding protein related peptide-receptive conformation has opened a new window to study (TAPBPR) associates with a broad range of partially folded MHC-I species the peptide-loading process in a range of detailed functional and inside the cell. Bimolecular fluorescence complementation and mechanistic studies (4, 6, 7). deep mutational scanning reveal that TAPBPR recognition is polarized Improper function of the antigen processing and presentation α toward the 2 domain of the peptide-binding groove, and depends on pathway confers susceptibility to diseases in a manner that is α the formation of a conserved MHC-I disulfide epitope in the 2 domain. highly dependent on the individual’s MHC-I haplotype and the Conversely, thermodynamic measurements of TAPBPR binding for a disease-relevant immunodominant peptides (8). Several stud- representative set of properly conformed, peptide-loaded molecules ies have provided key insights into the allelic preferences of suggest a narrower MHC-I specificity range. Using solution NMR, we chaperone interactions. It has been long established that some find that the extent of dynamics at “hotspot” surfaces confers TAPBPR MHC-I alleles require tapasin for proper peptide loading, recognition of a sparsely populated MHC-I state attained through a trafficking, and cell surface display, while others can intrinsically global conformational change. Consistently, restriction of MHC-I groove load peptides in tapasin knockouts (4, 6–10). In an extreme case plasticity through the introduction of a disulfide bond between the demonstrated by the HLA-B*44 alleles, a single amino acid BIOCHEMISTRY α1/α2 helices abrogates TAPBPR binding, both in solution and on a cellular membrane, while intracellular binding is tolerant of many Significance destabilizing MHC-I substitutions. Our data support parallel TAPBPR functions of 1) chaperoning unstable MHC-I molecules with broad The human population contains thousands of MHC-I alleles, allele-specificity at early stages of their folding process, and 2) editing showing a range of dependencies on molecular chaperones for the peptide cargo of properly conformed MHC-I molecules en route to loading of their peptide cargo, which are then displayed on the the surface, which demonstrates a narrower specificity. Our results cell surface for T cell surveillance. Using the chaperone TAPBPR suggest that TAPBPR exploits localized structural adaptations, both as a model, we combine deep mutagenesis with functional and near and distant to the peptide-binding groove, to selectively recog- biophysical data, especially solution NMR, to provide a complete nize discrete conformational states sampled by MHC-I alleles, toward view of the molecular determinants of chaperone recognition. editing the repertoire of displayed antigens. Our data provide significant evidence that localized protein motions define the intrinsic ability of MHC-I molecules to interact major histocompatibility complex | molecular chaperone | peptide with chaperones. The importance of MHC-I dynamics unifies our repertoire | NMR spectroscopy | peptide editing findings, with broad recognition of conformationally unstable, nascent MHC-I molecules becoming restricted to a smaller set of lass I major histocompatibility complex (MHC-I) molecules MHC-I alleles that retain relevant dynamic motions in their Cdisplay a diverse set of 8 to 14 residue peptide antigens to folded state. + CD8 cytotoxic T lymphocytes (1). This process provides a means for immune surveillance of the endogenous proteome to Author contributions: E.P. and N.G.S. designed research; A.C.M., C.A.D., S.A.O., J.P., J.S.T., detect invading pathogens or developing tumors. MHC-I molecules D.F.-S., H.C., S.T., and E.P. performed research; S.A.O., J.S.T., D.M., D.F.-S., H.C., and E.P. are extremely polymorphic, with thousands of known human contributed new reagents/analytic tools; A.C.M., C.A.D., S.A.O., J.P., J.S.T., D.F.-S., H.C., S.T., and E.P. analyzed data; and A.C.M., E.P., and N.G.S. wrote the paper. alleles, categorized in the HLA-A, -B, and -C classes. Specific interactions with highly polymorphic pockets along the peptide The authors declare no competing interest. binding groove (termed A- to F-pockets) define a repertoire of up This article is a PNAS Direct Submission. to 104 peptide antigens that can bind to each HLA protein (1). This open access article is distributed under Creative Commons Attribution-NonCommercial- NoDerivatives License 4.0 (CC BY-NC-ND). Proper folding of nascent MHC-I molecules and loading with Data deposition: Raw and processed Illumina sequencing data in this paper have been high-affinity peptides requires association with an invariant deposited in the Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih. light-chain, β2-microglobulin (β2m), and is facilitated by dedi- gov/geo (accession no. GSE128957). NMR assignments have been deposited into the Bi- cated molecular chaperones, tapasin, which is restricted within ological Magnetic Resonance Data Bank, http://www.bmrb.wisc.edu (accession nos. 27682 the peptide-loading complex (PLC), and the homologous, [H2-Ld], 27632 [HLA-A*01:01], and 27631 [HLA-A*02:01]). The atomic coordinates and structure factors for the P18-I10/H2-Dd Y84C-A139C/β2m complex have been deposited PLC-independent TAPBPR (TAP-binding protein related) (2, in the Protein Data Bank, www.pdb.org (PDB ID code 6NPR). 3). Furthermore, through the catalytic enhancement of peptide 1A.C.M. and C.A.D. contributed equally to this work. association and dissociation within the MHC-I groove, chaper- 2Present address: Structural Biology in Dementia, German Center for Neurodegenerative ones can influence the selection of immunodominant antigens by Diseases (DZNE), 37075 Göttingen, Germany. promoting the exchange of low- and intermediate-affinity for 3To whom correspondence may be addressed. Email: [email protected] or nsgourak@ high-affinity peptides (termed “peptide editing”) (2, 3). Further ucsc.edu. quality control and narrowing of the displayed antigen repertoire This article contains supporting information online at https://www.pnas.org/lookup/suppl/ along the trafficking pathway is accomplished through the doi:10.1073/pnas.1915562116/-/DCSupplemental. combined functions of TAPBPR and UDP-glucose:glycoprotein www.pnas.org/cgi/doi/10.1073/pnas.1915562116 PNAS Latest Articles | 1of12 Downloaded by guest on September 25, 2021 polymorphism at the F-pocket is sufficient to switch between low signal-to-noise, leading to false-positive BiFC signals for some chaperone-dependent and -independent peptide loading (11–13). protein–protein interactions (18). Thus, we coexpressed and Similarly, TAPBPR expression exhibits a marked effect on the coimmunoprecipitated TAPBPR with MHC-I in the absence of displayed repertoire of specific alleles (4). While structure-based VN and VC fusions. Unlike the CXCR4 control, all of the MHC-I modeling using the available cocrystal structures as templates can alleles bound TAPBPR, thereby independently confirming the provide some clues for the molecular basis of MHC-I allelic BiFC results using a different experimental method (SI Appendix, dependence of chaperone interactions, molecular dynamics Fig. S2). Together, these data suggest that TAPBPR associates (MD) simulations, and hydrogen deuterium exchange experiments with a broad range of MHC-I alleles inside the cell, potentially have further shown that chaperone-dependent and -independent through partially folded, empty MHC-I conformations within the alleles exhibit widespread differences in their dynamic profiles (14, endoplasmic reticulum or Golgi. 15). In particular, these experiments have suggested the rate of sampling of an “open” conformation confers chaperone recognition TAPBPR Recognizes a Partially Folded Epitope on MHC-I Molecules of different MHC-I alleles (16, 17). with Broad Specificity. Deep mutagenesis, in which directed evolu- Here, using the chaperone TAPBPR as a model, we apply a tion of a diverse library of sequences is tracked by next-generation range of complementary functional and biophysical experiments sequencing, has emerged as a powerful tool for interrogating how using fusion and recombinant proteins in a native cellular context protein amino acid sequence dictates structure and function in and in solution, respectively. We find that TAPBPR interacts living cells (19). The relative activities of thousands of mutations